Clinical Chemistry DM Vasudevan, Sreekumari S, Kannan Vaidyanathan, Geetha Damodaran K
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Glucose Homeostasis: Abnormalities and AssessmentCHAPTER 1

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REGULATION OF BLOOD GLUCOSE
The maintenance of glucose level in blood within narrow limits is a very finely and efficiently regulated system. This is important, because it is essential to have continuous supply of glucose to the brain. Brain has an obligatory requirement for glucose. RBC and renal medulla are also dependent on glucose for meeting their fuel needs. Factors maintaining the blood sugar are shown in Box 1.1 and Figure 1.3.
 
Post-prandial Regulation
Following a meal, glucose is absorbed from the intestine and enters the blood. The rise in the blood glucose level stimulates the secretion of insulin. The uptake of glucose by most extrahepatic tissues, except brain is dependent on insulin.3
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Fig. 1.1: Homeostasis of blood glucose
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Fig. 1.2: Blood glucose regulation in post-prandial state. Glucose level is initally high; then blood glucose level is lowered by tissue oxidation, glycogen synthesis and lipogenesis
4Moreover, insulin helps in the storage of glucose as glycogen or its conversion to fat (Figs 1.1 and 1.2.).
 
Regulation in Fasting State
Normally, 2 to 2½ hours after a meal, the blood glucose level falls to near fasting levels. It may go down further; but this is prevented by processes that contribute glucose to the blood.
For another 3 hours, hepatic glycogenolysis will take care of the blood sugar level. Thereafter, gluconeogenesis will take charge of the situation (Fig. 1.2).
Hormones like glucagon, epinephrine, glucocorticoids, growth hormone, ACTH and thyroxine will keep the blood glucose level from falling. They are referred to as anti-insulin hormones or hyperglycemic hormones. An overview of the regulatory mechanism is shown in Figure 1.3.
 
Normal Plasma Glucose Level
In normal persons, fasting plasma glucose value is 70–110 mg/dl (4.0–6.1 mmol/L).
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Fig. 1.3: Overview of regulation of blood sugar
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Fasting state means, glucose is estimated after an overnight fast (12 hours after the food) (post-absorptive state).
Following a meal, in a normal person the glucose level does not usually rise above 140 mg/dl due to prompt secretion of insulin. Hyperglycemia is harmful in the long run; while hypoglycemia even for a short while is dangerous, and may even be fatal.
The plasma values are slightly higher than whole blood glucose values because RBCs contain less water (73%) than plasma (93%).
For estimation of glucose, blood is collected using an anticoagulant (potassium oxalate) and an inhibitor of glycolysis (sodium fluoride). Fluoride inhibits the enzyme, enolase, and so glycolysis on the whole is inhibited. If fluoride is not added, cells will utilise glucose and false low value may be obtained. Capillary blood from finger tips may also be used for glucose estimation by strip method.
Estimation of glucose in blood is done by the enzymatic (GOD-POD) method. This is highly specific, giving ‘true glucose’ values (fasting 70–110 mg/dl). Present day autoanalysers can use only the enzymatic methods.
The glucose oxidase (GOD) is very specific; it converts glucose to gluconic acid and hydrogen peroxide. Then peroxidase (POD) converts the H2O2 into H2O and nascent oxygen. The oxygen oxidises a colourless chromogenic substrate (e.g., ortho di anisidine) to a coloured one; the colour intensity is directly proportional to concentration of glucose.
As a modification, the above GOD reaction mixture is immobilised on a plastic film (dry analysis). One drop of blood is placed over the reagent. The colour is developed within one minute. The intensity of dye is measured by reflectance photometry.
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Fig. 1.4: Oral Glucose Tolerance Test (OGTT)
The instrument is named as glucometer. It is useful for patients to have self-analysis at home. But the instrument is less accurate.
 
Sugar in Urine
Normally glucose is not excreted in urine. But if blood sugar is more than 180 mg/dl, urine contains glucose. The blood level of glucose above which glucose is excreted is called renal threshold (See Fig. 1.4).
 
ORAL GLUCOSE TOLERANCE TEST (OGTT)
 
Indications for OGTT
  1. Patient has symptoms suggestive of diabetes mellitus; but fasting blood sugar value is inconclusive (between 100 and 126 mg/dl).7
  2. During pregnancy, excessive weight gaining is noticed, with a past history of big baby (more than 4 kg) or a past history of miscarriage.
  3. To rule out benign renal glucosuria.
 
Contra-indications for OGTT
There is no indication for doing OGTT in a person with confirmed diabetes mellitus. It has no role in follow-up of diabetes. It is indicated only for the initial diagnosis.
 
Preparation of the Patient
The patient is instructed to have good carbohydrate diet for 3 days prior to the test. Further, diet containing about 30-50 g of carbohydrate should be taken on the evening prior to the test. This is important. Otherwise carbohydrates may not be tolerated even in a normal person.
Patient should avoid drugs likely to influence the blood glucose levels, for at least 2 days prior to the test. Patient should abstain from smoking during the test. Strenuous exercise on the previous day is to be avoided.
Patient should not take food after 8 PM the previous night. Should not take any breakfast. This is to ensure 12 hours fasting.
 
Conducting the Glucose Tolerance Test
At about 8 am, a sample of blood is collected in the fasting state. Urine sample is also obtained. This is denoted as the “0” hour sample.
Glucose Load Dose: The dose is 75 g anhydrous glucose (82.5 g of glucose monohydrate) in 250-300 ml of water. In 8order to prevent vomiting, patient is asked to drink it slowly (within about 5 minutes). Flavouring of the solution will also reduce the tendency to vomit. When the test is done in children, the glucose dose is adjusted as 1.75 g /kg body weight.
Sample Collection: In the classical procedure, the blood and urine samples are collected at 1/2 an hour intervals for the next 2½ hours. (Total six samples, including 0-hr sample). Glucose is estimated in all the blood samples. Urine samples are tested for glucose qualitatively.
Figure 1.4 represents the graph, when plasma glucose values are plotted on the vertical axis against the time of collection on the horizontal axis.
But the present WHO recommendation is to collect only the fasting and 2-hour post-glucose load samples of blood and urine. This is sometimes called mini-GTT. (Total 2 samples only). This is sufficient to get a correct assessment of the patient (see Box 1.2).
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Normal Values and Interpretations
In a normal person, fasting plasma glucose is 70–110 mg/dl. Following the glucose load, the level rises and reaches a peak within 1 hour and then comes down to normal fasting levels by 2 to 2½ hours. None of the urine sample shows any evidence of glucose.
 
Diagnostic Criteria for Diabetes Mellitus
  1. If the fasting plasma sugar is more than 126 mg /dl, on more than one occasion (Table 1.1).
  2. Or, if 2-hr post-glucose load value of OGTT is more than 200 mg /dl (even at one occasion).
  3. Or, if both fasting and 2-hr values are above these levels, on the same occasion.
  4. If the random plasma sugar level is more than 200 mg/dl, on more than one occasion. Diagnosis should not be based on a single random test alone; it should be repeated.
Table 1.1   The plasma sugar levels in OGTT in normal persons and in diabetic patients
Normal persons
Criteria for diagnosing diabetes
Criteria for diagnosing I.G.T
Fasting
< 110 mg/dl
<(6.1mmol/L)
> 126 mg/dl
>(7.0 mmol/L)
110 to
126 mg/dl
1 hr (peak)
after glucose
< 160 mg/dl
<(9 mmol/L)
Not
prescribed
Not
prescribed
2 hr after
glucose
< 140 mg/dl
<(7.8mmol/L)
> 200 mg/dl
>(11.1mmol/L)
140 to
199mg/dl
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Abnormal GTT Curve
 
1. Impaired Glucose Tolerance (IGT)
It is otherwise called as Impaired Glucose Regulation (IGR). Here blood sugar values are above the normal level, but below the diabetic levels. In IGT, the fasting plasma glucose level is between 110 and 126 mg/dl and 2-hour post-glucose value is between 140 and 200 mg/dl (see Table 1.1). Such persons need careful follow up because IGT progresses to frank diabetes at the rate of 2% patients per year.
 
2. Impaired Fasting Glycemia (IFG)
In this condition, fasting plasma sugar is above normal (between 110 and 126 mg/dl); but the 2-hour post-glucose value is within normal limits (less than 140 mg/dl). These persons need no immediate treatment; but are to be kept under constant check up.
 
3. Gestational Diabetes Mellitus (GDM)
This term is used when carbohydrate intolerance is noticed, for the first time, during a pregnancy. A known diabetic patient, who becomes pregnant, is not included in this category. GDM is associated with an increased incidence of neonatal mortality. After the child birth, the women should be re-assessed.
 
4. Alimentary Glucosuria
Here the fasting and 2-hr values are normal; but an exaggerated rise in blood glucose following the ingestion of glucose is seen. This is due to an increased rate of absorption 11of glucose from the intestine. This is seen in patients after a gastrectomy or in hyperthyroidism.
 
5. Renal Glucosuria
Normal renal threshold for glucose is 175–180 mg/dl. If blood sugar rises above this, glucose starts to appear in urine. When renal threshold is lowered (renal tubular reabsorption is lowered), glucose is excreted in urine, even if blood sugar is within normal limits. This is called renal glycosuria.
 
REDUCING SUBSTANCES IN URINE
The excretion of reducing substances in urine is detected by a positive Benedict's test. Such conditions together are sometimes called as “mellituria”. These conditions are enumerated in Table 1.2. When reducing sugars are excreted in urine, the condition is referred to as glycosuria.
Table 1.2   Differential diagnosis of reducing substances in urine
1. Glucosuria
  • 1-a. Diabetes Mellitus
  • 1-b. Transient glucosuria
  • 1-c. Alimentary glucosuria
  • 1-d. Renal glucosuria
2. Fructosuria
  • 2-a. Deficiency of fructokinase
  • 2-b. Fructose intolerance (aldolase B deficiency)
3. Lactosuria
4. Galactosuria (Deficiency of galactose-1-phosphate uridyl transferase)
5. Pentosuria (xylulosuria)
6. Non-carbohydrate reducing substances
  • 6-a. Glucuronides, salicylate
  • 6-b. Ascorbic acid (vitamin C)
  • 6-c. Homogentisic acid
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To denote the excretion of specific sugars the suffix ‘uria’ is added to the name of the sugar, e.g. glucosuria, fructosuria, lactosuria. Glucosuria means glucose in urine; glycosuria means any sugar in urine. Since glucose is the most common reducing sugar excreted in urine, the term glycosuria is often (though incorrectly) used to denote the excretion of glucose.
 
Hyperglycemic Glucosuria
  1. When blood glucose level exceeds the renal threshold (175—180 mg/dl), glucose is excreted in urine. Diabetes mellitus is the most common cause.
  2. Transient glucosuria: It may occur in some people due to emotional stress. Excessive secretion of anti-insulin hormones like cortisol (anxiety) and thyroid hormone may cause glucosuria. Once the stress is removed, the glucosuria disappears.
  3. Renal glucosuria
  4. Alimentary glucosuria; both are described under glucose tolerance test.
 
Lactosuria
It is the second most common reducing sugar found in urine. It is observed in the urine of normal women during 3rd trimester of pregnancy and lactation. The condition is harmless. In pregnancy, it is important to distinguish lactosuria from glucosuria when gestational diabetes mellitus is suspected.
 
Pentosuria
Essential pentosuria is characterised by the excretion of L-xylulose in urine due to deficiency of enzyme xylulose 13dehydrogenase. It is a harmless condition. Pentosuria may occur due to ingestion of cherries, berries and plums.
 
Non-carbohydrate Reducing Compounds
Glucuronides: Many drugs, e.g. isonictonic acid, para-amino salicylate, penicillin, cephaloxin, nalidixic acid are excreted as conjugates of glucuronic acid. In alkaline conditions, the glucuronic acid is released, which is a powerful reducing agent. So, Benedict's test will be positive.
Ascorbic Acid: Ascorbic acid or vitamin C is a very common ingredient of many tonics. Persons taking such tonics will excrete ascorbic acid in urine. It is a powerful reducing agent. This may cause confusion, as the Benedict's test is positive in such normal individuals.
 
Benedict's Test
About 0.5 ml of urine is boiled with 5 ml Benedict's reagent for 2 minutes (or kept for 2 minutes in water bath which is already boiling). The formation of a precipitate is observed on cooling.
The test is semi-quantitative and the colour of the precipitate roughly parallels the concentration of reducing sugar. Blue colour indicates the absence of sugar in urine. The green precipitate means 0.5%; yellow (1%); orange (1.5%) and red indicates 2% or more of sugar (1% means 1 g per 100 ml).
Any reducing sugar will give a positive Benedict's test. So differentiation of various sugars which may be present in urine has practical importance.14
 
Biosynthesis of Insulin
Insulin is a protein synthesised and secreted by the beta- cells of the islets of Langerhans of the pancreas. The proinsulin with 86 amino acids is cleaved; the C-peptide or connecting peptide with 33 amino acids is removed. Insulin with 51 amino acids is thus formed (Fig.1.5).
Normal insulin level in blood is 5–15 micro units/ml. Proinsulin contributes 5 to 10% of the total insulin measured in plasma. Proinsulin has about one-third biological activity as that of insulin. Insulin and C-peptide are synthesised and secreted in equimolar quantities. Measurement of C-peptide is an index of rate of secretion of insulin. Insulin and C-peptide estimations are not commonly done in routine clinical practice; but are done for experimental purposes.
 
Factors Increasing Insulin Secretion
 
1. Glucose
Glucose is the major stimulant of insulin secretion. As blood glucose level increases, the insulin secretion also correspondingly increases.
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Fig. 1.5: Insulin biosynthesis
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The beta cells have GluT 2 receptors, through which glucose is absorbed. This is oxidised, so that more ATP is made available. ATP stimulates an ATP Binding Cassette protein (ABC protein). Simultaneously potassium channels are closed and calcium channels are opened. Increased intracellular calcium causes the insulin secretion.
 
2. Gastrointestinal Hormones
Insulin secretion is enhanced by secretin, pancreozymin and gastrin. After taking food, these hormones are increased.
Incretin hormones: Glucose dependent insulinotropic polypeptide (GIP, 41 amino acids) and Glucagon like peptide 1 (GLP-1, 31 amino acids) are involved in the release of insulin following nutrient entry into stomach. They are both secreted by specialised cells in the gastro-intestinal tract and have receptors located in islet cells. They have a very short half life. For treatment of diabetes, new drugs are being developed either to mimic or to target these hormones.
  • Proteins and amino acids: Leucine and arginine are stimulants.
  • Parasympathetic and beta-adrenergic stimulation.
  • Glucagon and growth hormone.
  • Drug, Tolbutamide.
 
Factors decreasing the Insulin Secretion
Epinephrine: During stressful conditions and during exercise, adrenal medulla releases adrenaline. This suppresses insulin release, and at the same time, mobilises glucose from liver for energy purpose. Alpha adrenergic stimulation also reduces insulin.16
 
Physiological Actions of Insulin
 
1. Uptake of Glucose by Tissues
Insulin facilitates the membrane transport of glucose. Facilitated diffusion of glucose in muscle is enhanced by insulin. In diabetes mellitus, the transporter, GIuT4 is reduced. However, glucose uptake in liver (by GluT2) is independent of insulin.
 
2. Utilisation of Glucose
Glycolysis is stimulated by insulin. (Table 1.3).
 
3. Hypoglycemic Effect
Insulin lowers the blood glucose level by promoting utilisation and storage. Insulin inhibits gluconeogenesis and glycogenolysis. So, blood glucose level is lowered. (Table 1.3).
 
4. Lipogenesis
Lipogenesis is favoured by providing more acetyl CoA by pyruvate dehydrogenase reaction and also by increasing the availability of NADPH.
 
5. Anti-lipolytic Effect
Insulin inhibits lipolysis in adipose tissue due to inhibition of hormone sensitive lipase. The increased level of FFA in plasma in diabetes is due to the loss of this inhibitory effect on lipolysis.
 
6. Anti-ketogenic Effect
Insulin depresses HMG CoA synthase and so ketogenesis is decreased.
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Table 1.3   Comparison of action of insulin and antiinsulin hormones
Metabolism
Key enzymes
Insulin
Glucagon
Gluco-corticoids
Growth hormone
Glycolysis
GK, PFK and PK
Stimulation
Inhibition
Gluconeogenesis
PEPCK, G6 Pase,
Inhibition
Stimulation
Stimulation
Stimulation
F-bisphosphatase
Glycogen synthesis
Glycogen synthase
Activation
Inhibition
Glycogenolysis
Phosphorylase
Inactivation
Activation
Lipolysis
Hormone sensitive lipase
Inhibition
Stimulation
stimulation
Stimulation
Ketogenesis
Carnitine acyl transferase
Inhibition
Stimulation
Stimulation
Protein breakdown
Transaminases
Inhibition
Stimulation
Protein synthesis
Anabolism
Catabolism
Anabolism
Blood sugar level
Decreases
Increases
Increases
Increases
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Moreover, in presence of insulin, acetyl CoA is completely utilised in the citric acid cycle, because oxaloacetate generated from glucose is available in plenty. Insulin also favours fatty acid synthesis from acetyl CoA. All these factors reduce the availability of acetyl CoA, so that production of ketone bodies is reduced.
 
GLUCAGON
Glucagon is the most potent hyperglycemic hormone. It is anti-insulin in nature. Therefore, the net effect is decided by the insulin-glucagon ratio. Glucagon is mainly glycogenolytic. Liver is the primary target for the glycogenolytic effect of glucagon. It simultaneously depresses glycogen synthesis. Gluconeogenesis is also favored by glucagon. Glucagon increases plasma free fatty acid level. In adipose tissue glucagon favours beta-oxidation. Ketogenesis is favoured. (Table 1.3).
The effects of other anti-insulin hormones are summarised in Table 1.3.
 
DIABETES MELLITUS
Diabetes mellitus is a metabolic disease due to absolute or relative insulin deficiency. Diabetes mellitus is a common clinical condition. About 10% of the total population, and about 1/5th of persons above the age of 50, suffer from this disease. It is a major cause for morbidity and mortality. Insulin deficiency leads to increased blood glucose level. In spite of this high blood glucose, the entry of glucose into the cell is inefficient. Hence all cells are starved for glucose.
  1. Type 1 Diabetes Mellitus: (Formerly known as Insulin-dependent diabetes mellitus; IDDM). About 5% of total 19diabetic patients are of type 1. Here circulating insulin level is deficient. It is subclassified as : a) immune mediated and b) idiopathic.
  2. Type 2 Diabetes Mellitus: (Formerly known as non-insulin dependent diabetes mellitus; NIDDM). Most of the patients belong to this type. Here circulating insulin level is normal or mildly elevated or slightly decreased, depending on the stage of the disease. This type is further classified as: a) obese; b) non-obese and c) maturity onset diabetes of young (MODY).
  3. Diabetic prone states: (a) Gestational diabetes mellitus (GDM); (b) Impaired glucose tolerance (IGT); (c) Impaired fasting glycemia (IFG)
  4. Secondary to other known causes: (a) endocrinopathies (Cushing's disease, thyrotoxicosis, acromegaly); (b) drug induced (steroids, beta blockers, etc.); (c) pancreatic diseases (chronic pancreatitis, fibro calculus pancreatitis, hemochromatosis, cystic fibrosis).
 
Metabolic Syndrome (MetS) and Polycystic Ovary Syndrome (PCOS)
They have overlapping features. The common factors are insulin resistance and obesity. In PCOS there is hyperandrogenism. Those with PCOS have an increased risk for coronary vascular disease as in patients with MetS. PCOS may be corrected to some extent by lifestyle modification during the adolescent age by weight reduction and exercise. A higher prevalence of PCOS is seen in women with Type 2 diabetes mellitus. Overweight adolescents with PCOS are at increased risk of developing impaired glucose tolerance and Type 2 diabetes mellitus.20
 
Type 1 Diabetes Mellitus
It is due to decreased insulin production. Circulating insulin level is very low. These patients are dependent on insulin injections. Onset is usually below 30 years of age, most commonly during adolescence. They are more prone to develop ketosis. Circulating antibodies against insulin is seen in 50% cases, and antibodies against islet cell cytoplasmic proteins are seen in 80% cases.
 
Type 2 Diabetes Mellitus
About 95% of the patients belong to this type. The disease is due to the decreased biological response to insulin, otherwise called insulin resistance. So there is a relative insulin deficiency. Type 2 disease is commonly seen in individuals above 40 years. These patients are less prone to develop ketosis. About 60% of patients are obese. These patients have high plasma insulin levels.
The maturity onset diabetes of young (MODY) is due to defective glucokinase (GK). This mutation produces relative insulin deficiency by increasing the threshold for glucose induced insulin secretion.
 
Metabolic Syndrome
It is characterized by i) Abdominal obesity; ii) Hyper-triglyceridemia, low HDL cholesterol; iii) Elevated blood pressure and iv) Insulin resistance or decreased glucose intolerance. The body cannot properly use glucose even in presence of normal insulin level. In other words, body cannot use insulin efficiently. Therefore, the metabolic syndrome is also called the insulin resistance syndrome. People with the 21metabolic syndrome are at increased risk of coronary heart disease and type 2 diabetes. The metabolic syndrome has become increasingly common in the developing countries.
Acquired factors, such as excess body fat and physical inactivity, can elicit insulin resistance and the metabolic syndrome in these people. Most people with insulin resistance have abdominal obesity. Criteria for diagnosis of metabolic syndrome are:
  1. Elevated waist circumference: For men equal to or greater than 40 inches (102 cm) and for women, equal to or greater than 35 inches (88 cm).
  2. Elevated triglycerides: Equal to or greater than 150 mg/dL
  3. Reduced HDL (“good”) cholesterol: For men, less than 40 mg/dL; for women, < 50 mg/dL
  4. Elevated blood pressure: Equal to or greater than 130/85 mm Hg
  5. Elevated fasting glucose: Equal to or greater than 100 mg/dL.
Management of Metabolic Syndrome includes: Weight reduction to achieve a desirable weight. Moderate exercise every day. Reduced intake of saturated fats, trans fatty acids and cholesterol.
 
Metabolic Derangements in Diabetes
 
1. Derangements in Carbohydrate Metabolism
Insulin deficiency decreases the uptake of glucose by cells. The insulin dependent enzymes are also less active. Net effect is an inhibition of glycolysis and stimulation of gluconeogenesis leading to hyperglycemia.22
 
2. Derangements in Lipid Metabolism
Fatty acid breakdown leads to high FFA levels of plasma and consequent fatty liver. There is excess acetyl CoA which is diverted to production of ketone bodies, leading to ketogenesis. This tendency is seen more in Type 1 disease. There is hyperlipidemia, especially an increase in NEFA, TAG and cholesterol in plasma.
 
3. Derangement in Protein Metabolism
Increased breakdown of proteins and amino acids for providing substrates for gluconeogenesis is responsible for muscle wasting.
 
Clinical Presentations in Diabetes Mellitus
 
Cardinal Symptoms
  1. When the blood glucose level exceeds the renal threshold glucose is excreted in urine (glucosuria).
  2. Due to osmotic effect, more water accompanies the glucose (polyuria).
  3. To compensate for this loss of water, thirst centre is activated, and more water is taken (polydypsia).
  4. To compensate the loss of glucose and protein, patient will take more food (polyphagia).
  5. The loss and ineffective utilisation of glucose leads to breakdown of fat and protein. This would lead to loss of weight. Important differential diagnosis for weight loss are diabetes mellitus, tuberculosis, hyperthyroidism, cancer and AIDS.
  6. Often the presenting complaint of the patient may be chronic recurrent infections such as boils, abscesses, etc. 23Any person with recurrent infections should be investigated for diabetes. In India, tuberculosis is commonly associated with diabetes.
 
Acute Metabolic Complications
 
KETOSIS
Normally the blood level of ketone bodies is less than 1 mg/dl and only traces are excreted in urine (not detectable by usual tests). But when the rate of synthesis exceeds the ability of extrahepatic tissues to utilise them, there will be accumulation of ketone bodies in blood. This leads to ketonemia, excretion in urine (ketonuria) and smell of acetone in breath. All these three together constitute the condition known as ketosis.
 
Causes for Ketosis
  1. Diabetes Mellitus: Untreated diabetes mellitus is the most common cause for ketosis. Even though glucose is in plenty, the deficiency of insulin causes accelerated lipolysis and more fatty acids are released into circulation. Oxidation of these fatty acids increases the acetyl CoA pool. Enhanced gluconeogenesis restricts the oxidation of acetyl CoA by TCA cycle, since availability of oxaloacetate is less.
  2. Starvation: In starvation, the dietary supply of glucose is decreased. Available oxaloacetate is channelled to gluconeogenesis. The increased rate of lipolysis is to provide alternate source of fuel. The excess acetyl CoA is converted to ketone bodies. The high glucagon favours ketogenesis. Hyperemesis (vomiting) in early pregnancy may also lead to starvation-like condition and may lead to ketosis.24
In both diabetes mellitus and starvation, the oxaloacetate is channelled to gluconeogenesis; so the availability of oxaloacetate is decreased. Hence acetyl CoA cannot be fully oxidised in the TCA cycle. Oxaloacetate is diverted for gluconeogenesis; then citric acid cycle cannot function optimally. Thus, on the one hand, acetyl CoA is generated in excess, on the other hand, its utilisation is reduced. This excess acetyl CoA is channelled into ketogenic pathway. Thus, on the one hand, acetyl CoA is generated in excess, on the other hand, its utilisation is reduced. This excess acetyl CoA is channelled into ketogenic pathway. (See Fig. 1.6).
zoom view
Fig. 1.6: Metabolic derangements in Diabetes Mellitus
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Consequences of Ketosis
Metabolic acidosis: Acetoacetate and beta-hydroxy butyrate are acids. When they accumulate, metabolic acidosis results. (See Chapter 8).
Reduced buffers: The plasma bicarbonate is used up for buffering of these acids.
Kussmaul's respiration: Patients will have typical acidotic breathing due to compensatory hyperventilation.
Smell of acetone in patient's breath.
Osmotic diuresis induced by ketonuria may lead to dehydration.
Sodium loss: The ketone bodies are excreted in urine as their sodium salt, leading to loss of cations from the body.
Dehydration: The sodium loss further aggravates the dehydration.
Coma: Hypokalemia, dehydration and acidosis are contributing for the lethal effect of ketosis.
 
Diagnosis of Ketosis
The presence of ketosis can be established by the detection of ketone bodies in urine by Rothera's test. Supportive evidence may be derived from estimation of serum electrolytes, acid–base parameters and glucose estimation.
Rothera's Test: Saturate 5 ml of urine with solid ammonium sulphate. Add a few drops of freshly prepared sodium nitroprusside followed by 2 ml of liquor ammonia along the sides of the test tube. Development of a purple ring indicates 26the presence of ketone bodies in urine. Strip tests based on the same principle are also available.
 
Differential Diagnosis of Ketosis
The urine of a patient with diabetic keto acidosis will give positive Benedict's test as well as Rothera's test. But in starvation ketosis, Benedict's test is negative, but Rothera's test will be positive.
 
Management of Ketoacidosis
  1. Parenteral administration of insulin and glucose by intravenous route to control diabetes. When glucose and insulin are given intravenously, potassium is trapped within the cells. Hence the clinician should always monitor the electrolytes.
  2. Intravenous bicarbonate to correct the acidosis.
  3. Correction of water imbalance.
  4. Correction of electrolyte imbalance.
  5. Treatment of underlying precipitating causes.
 
Hyperosmolar Nonketotic Coma
It can result due to elevation of glucose to very high levels (900 mg/dl or more). This would increase the osmolality of extracellular fluid (ECF). Osmotic diuresis leads to water and electrolyte depletion. The coma results from dehydration of cerebral cells due to hypertonicity of ECF.
 
Chronic Complications of Diabetes Mellitus
  1. Vascular Diseases: Atherosclerosis in medium sized vessels, plaque formation and consequent intravascular 27thrombosis may take place. If it occurs in cerebral vessels, the result is paralysis. If it is in coronary artery, myocardial infarction results. In the case of small vessels, the process is called micro-angiopathy, which may lead to diabetic retinopathy and nephropathy.
  2. Complications in Eyes: Early development of cataract of lens is due to the increased rate of sorbitol formation, caused by the hyperglycemia. Retinal microvascular abnormalities lead to retinopathy and blindness.
  3. Neuropathy: Peripheral neuropathy with paresthesia is very common. Decreased glucose utilisation and its diversion to sorbitol in Schwann cells may be cause for neuropathy. Neuropathy may lead to risk of foot ulcers and gangrene. Hence care of the feet in diabetic patients is important.
 
Laboratory Investigations in Diabetes
 
1. Blood Glucose Level:
Random blood sugar estimation and oral glucose tolerance tests are used for the diagnosis (Table 1.1). For monitoring a diabetic patient, periodic check of fasting and postprandial blood glucose are to be done at least once in 3 months. Blood glucose level has to be maintained within the normal limits.
 
2. Complete lipid profile
Total cholesterol, triglycerides, HDL and LDL cholesterol levels may be done once in six months (Chapter 2).28
 
3. Kidney Function Tests
Blood urea and serum creatinine may be done at least twice an year.
 
4. Micro Albuminuria and Frank Albuminuria
Presence of albumin (30 to 300 mg/day) in urine is known as micro-albuminuria. It is a predictor of progressive renal damage. Albumin more than 300 mg/day indicates overt diabetic nephropathy. Microalbuminuria is to be checked atleast once in an year.
 
5. Glycated Hemoglobin
The best index of long term control of blood glucose level is measurement of glycated hemoglobin or glyco-hemoglobin. Enzymatic addition of any sugar to a protein is called “glycosylation”, while non-enzymatic process is termed “glycation”.
When there is hyperglycemia, proteins in the body may undergo glycation. It is a non-enzymatic process. Glucose forms a Schiff base with the N-terminal amino group of proteins. When once attached, glucose is not removed from hemoglobin. Therefore, it remains inside the erythrocyte, throughout the life span of RBCs. (120 days) (Fig. 1.7).
zoom view
Fig. 1.7: Glycation is parallel to the blood glucose
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The glycated hemoglobins are together called HbA1 fraction. Out of this 80% molecules are HbA1c, where glucose is attached to the N-terminal valine of beta chain of hemoglobin. The determination of glycated hemoglobin is not for diagnosis of diabetes mellitus; but only for monitoring the response to treatment.
Normally the level of Hb A1c is about 4–7%. But in diabetic patients, the value is increased to 8–12%.
The HbA1c level reveals the mean glucose level over the previous 10 -12 weeks. It is unaffected by recent food intake or recent changes in blood sugar levels. An elevated glycohemoglobin indicates poor control of diabetes mellitus. The risk of retinopathy and renal complications are proportionately increased with elevated glycated hemoglobin value. Reduction in 1% of glycoHb will decrease long term complications to an extent of 30%.
The estimation should be done atleast every 3 months in all patients on treatment. It is preferable to do the test every month, so that the effect of the treatment could be monitored closely. GlycoHb value below 7% indicates adequate control of diabetic state.
 
7. Other Glycated Proteins (Fructosamines)
Along with other proteins, albumin is also glycated in diabetes mellitus. Glycated albumin is more correctly called as fructosamino albumin. As half-life of albumin is about 20 days, gluco-albumin concentration reflects the glucose control over a recent past, for a period of last 2-3 weeks. Estimation of serum fructosamine is preferred in gestational diabetes mellitus.30
Glycation of other tissues proteins (lens proteins laminin, kidney glomerular membrane) are also reported. This will lead to functional abnormality of the cells or tissues. Glycation of DNA and DNA-proteins are also causing functional alterations.
 
Management of Diabetes Mellitus
  1. Diet and Exercise: This is the first line of treatment. A diabetic patient is advised to take a balanced diet with high protein content, low calories, devoid of refined sugars and low saturated fat, adequate PUFA, low cholesterol and sufficient quantities of fiber. Vegetables are the major sources of minerals, vitamins and fiber.
  2. Oral hypoglycemic agents: They are mainly of two types; sulphonyl urea and biguanides. They are mainly used in Type 2 diabetes.
  3. Insulin injections: Insulin is the drug of choice in Type 1 disease. It is also used in Type 2 disease, where oral drugs are not sufficient. Development of human insulin (produced by recombinant DNA technology) has revolutionised the management of diabetes mellitus.
  4. Prevention of complications.
 
Hypoglycemia
Hyperglycemia causes harm; but hypoglycemia is fatal. A fall in plasma glucose less than 50 mg/dl is life-threatening. Causes of hypoglycemia are:
  1. Overdose of insulin: This is the most common cause. The differentiation of hypoglycemic coma from hyperglycemic 31coma (ketosis) is important, since treatment is exactly opposite. The diagnosis is mainly based on blood glucose estimation.
  2. Post-prandial hypoglycemia: 2-3 hours after a meal, transient hypoglycemia is seen in some persons. This is due to over-secretion of insulin.
  3. Insulinoma: Insulin secreting tumors are rare.